Successful hematopoietic transplantation relies on the capacity of intravenously infused hematopoietic stem cells (HSC) to home to and engraft bone marrow. This ability to home and migrate is central not only to the effective functioning of hematopoietic cells, but also to the development of the hematopoietic system. During vertebrate development, the site of hematopoieis moves from the yolk sac of the embryo to the embryo dorsal aorta to the fetal liver, then to the fetal spleen and finally to the bone marrow. Thus the HSC at each of these developmental stages must be able to migrate and home to location(s) that support their ability to self-renew and differentiate. Under this grant, we have made several surprising findings. First, we identified the homing receptors and chemokine receptors used by HSC to home from blood to marrow hematopoiesis sites. Next, we showed that fully competent, long term self-renewing HSC constitutively migrate through the blood and are capable of homing to niches, even in a parabiotic partner, and there establish long term hematopoiesis. Although ~100 HSC are in the blood of a mouse at any given time, their residence time in the blood, <5', is consistent with a daily flux of >104 HSC/mouse/day from marrow to blood to marrow. We now confirm that at any moment, up to 0.5% of marrow engraftment sites (niches) are open at a time, perhaps those vacated by the emigrating cells; these can accept donor HSC without conditioning. In SCID mice (and perhaps humans), single or repetitive (timed to match the opening of another 0.5% of niches) HSC infusions into unconditioned hosts gives significant engraftment and cures the immunodeficiency. In this grant we plan to pursue the specificity and nature of these engraftment sites and to better understand barriers to engraftment. We have developed an assay for the in vivo ectopic development and maintenance of bone marrow niches that will allow purification of the cells responsible for both niche development and niche maintenance; and have shown that modification of expression of particular proteins in these precursors can modify niche development and function. We will address the following specific aims: 1) To characterize the kinetics, magnitude, and therapeutic potential of HSC homing to and from BM sites under steady state conditions; 2) To establish whether blockade of macrophage phagocytosis by CD47 enables mouse and human HSC homing to hematopoietic microenvironments and 3) To isolate and characterize HSC-supportive niche generating cells.